Aims: This study aimed to investigate the corneal biomechanical changes and topographic outcomes of accelerated transepithelial corneal cross-linking (ATE-CXL) in pediatric progressive keratoconus.Methods: In this prospective longitudinal study, 31 eyes of 28 pediatric patients with keratoconus  undergoing ATE-CXL (epithelium-on procedure with 45 mW/cm2 for 320 s) were included. Corvis ST was used to measure dynamic corneal response parameters at baseline and at 12 month after ATE-CXL. Corneal keratometry and corneal thickness were measured using Pentacam pre-operatively and 1, 6, and 12 month post-operatively.Results: No serious complications occurred during or after ATE-CXL. The maximum keratometry values were 60.10 ± 7.51 D pre-operatively and 61.42 ± 8.92, 61.17 ± 7.96, and 60.02 ± 7.58 D at 1, 6, and 12 month after ATE-CXL (P > 0.05), respectively. Corneal thickness remained stable during the 12-month follow-up (P > 0.05). At post-operative 12 month, first applanation time (P < 0.001), first applanation length (P = 0.004), second applanation velocity (P = 0.014), highest concavity time (P = 0.022), and radius of curvature at highest concavity (P = 0.031) increased significantly. The value of stiffness parameter at first applanation was significantly increased from 57.70 ± 27.57 pre-operatively to 63.36 ± 27.09 at 12 months after ATE-CXL (P = 0.018).Conclusions: ATE-CXL is safe and effective in stabilizing the progression of pediatric keratoconus. Changes in corneal biomechanical response consistent with stiffening following ATE-CXL were observed in pediatric patients with keratoconus. ATE-CXL is safe and effective in stabilizing the progression and the corneal biomechanical properties of pediatric keratoconus.Keratoconus is a bilateral progressive non-inflammatory ectatic corneal dystrophy characterized by thinning and steepening of the paracentral cornea , which u2 for 30 min) in the corneas of pediatric patients. Accelerated transepithelial corneal cross-linking   is considered an effective treatment for halting or reducing the progression of keratoconus with increasing corneal biomechanical stiffness. The majority of published studies –7 have dr 320 s) –11 has sr 320 s) , 11] thein vivo.Abnormal corneal biomechanical behavior is the most probable etiologic factor for keratoconus, thereby resulting in corneal morphologic changes . AccordiSeveral published studies have reported the use of Corvis ST in assessing the biomechanical properties of the cornea and in the diagnosis and treatment of keratoconus , 16. A pIn this prospective study, we enrolled pediatric patients (aged 8–17 years) who were diagnosed with progressive keratoconus at the Eye and ENT Hospital of Fudan University in Shanghai, China. Evidence of progressive keratoconus included an increase in the maximum keratometry (Kmax) value or astigmatism >1 D in 1 year. Patients were excluded from the study if they had any of the following: stromal scarring in the cornea; worn rigid gas permeable lenses or soft contact lenses more than 4 or 2 weeks, respectively; a history of other ocular diseases that could affect corneal morphology; previous ocular surgeries; or allergy to riboflavin. We analyzed 31 eyes  of 28 patients with a mean age of 14.35 ± 2.68 years. The baseline characteristics of the study population are shown in This study was approved by the Ethics Committee of the Eye and ENT Hospital of Fudan University and was carried out following the tenets of the Declaration of Helsinki. One parent or legal guardian of each subject was provided with informed consent after a detailed explanation of the procedure prior to treatment.via Pentacam . All measurements with the Pentacam were operated by the same experienced technicians. The data were collected pre-operatively and at 1, 6, and 12 month after ATE-CXL.The central corneal thickness (CCT), thinnest corneal thickness (TCT), apex thickness (AT), steepest meridian keratometry (K1), flattest meridian keratometry (K2), and Kmax values were obtained DCR parameters were measured by the Corvis ST. The Corvis ST can capture series images of cornea by using the ultra-high-speed Scheimpflug camera while consistent air puff deforms the cornea. The definitions of the DCR parameters are listed in 2 in pulsed mode . A balanced salt solution was applied to protect the cornea from dehydration during the irradiation. At the end of the procedure, the eye was patched with a bandage contact lens, and the contact lens was removed after 3 days until epithelial healing was complete. Post-operatively, 0.1% fluorometholone was prescribed seven times per day, and the frequency was gradually tapered down over 2 weeks. Additionally, topical antibiotic (levofloxacin) and artificial tears were prescribed for four times per day for a week and month, respectively.All operations were performed by the same surgeon (X Zhou). ATE-CXL was initiated under topical anesthesia using oxybuprocaine hydrochloride eye drops. The cornea was treated by using ParaCel solution  in the trephine  for 4 min, and then Vibex Xtra solution  was dripped into the trephine for 6 min. After that, the cornea was rinsed with a balanced salt solution. Subsequently, the cornea was then exposed to 370 nm ultraviolet-A (UVA) light with the Avedro's KXL System  for 5 min and 20 s at an irradiance level of 45 mW/cmpost-hoc comparisons. Inter-eye correlation was adjusted using a linear mixed model with random intercept to compare preoperative and post-operative parameters. Since certain DCR parameters were influenced by intraocular pressure (IOP)  and SAS . The results were analyzed using the Wilcoxon rank-sum test and repeated measures analyses of variance with Bonferroni-adjusted re (IOP) , it was All surgical procedures were completed successfully without any intraoperative or post-operative complications.P > 0.05) changes in K1, K2, and Kmax values in comparison to preoperative values at each follow-up time point.Pre- and post-operative corneal keratometry values are presented in P > 0.05) changes were found for CCT, TCT, and AT values during the 1-year follow-up.P < 0.001, P = 0.004, P = 0.014, P = 0.022, and P = 0.031, respectively) in 1 year after ATE-CXL. Furthermore, The SP-A1 value significantly increased from 57.70 ± 27.57 preoperatively to 63.36 ± 27.09 at 1 year post-operatively (P = 0.018). There were no significant differences in other parameters between the preoperative and post-operative time points (P > 0.05).The results of the corneal biomechanical properties assessment using the Corvis ST are illustrated in CXL is an effective treatment widely used to treat keratoconus. It is important to evaluate the efficacy of CXL by examination after the procedure, especially for pediatric patients with keratoconus because of the fast visual-developing period and the fact that any pathogenic effects may lead the irreversible vision loss. Corvis ST can be used to provide a series of DCR parameters in the diagnosis and treatment assessment of patients with keratoconus. In this study, we are the first to investigate the changes of corneal biomechanical properties in pediatric patients with progressive keratoconus after ATE-CXL.Our results showed that the eyes remained stable with respect to Kmax 1 year after ATE-CXL when compared to the preoperative assessment, which may indicate that ATE-CXL is an effective treatment for halting the progression of pediatric keratoconus. Or et al.  and EissThe obtained results suggested no significant changes in CCT, TCT, and AT 1 year after ATE-CXL, which indicated the efficacy of stabilizing the corneal thickness in pediatric progressive keratoconus. Previous studies , 7, 21 aIn this study, no intraoperative or post-operative complications were observed during the follow-up, which indicated that ATE-CXL can be safely used to treat pediatric patients. It has been reported that –24 complOur comparative analysis of the corneal biomechanical parameters obtained before and after ATE-CXL showed that AT1 and AL1 increased significantly during 1-year follow-up. Vinciguerra et al.  reportedThe deformation amplitude of a stiffer cornea is lower after being impacted by the pulse air, so the SP-A1 value is higher as detected by Corvis ST. Therefore, a healthy cornea shows a higher SP-A1 value compared with those with keratoconus . AnotherThere were some limitations in this study. One was a relatively low number of patients, and another was the lack of a control group undergoing C-CXL. In addition, 1-year results of ATE-CXL is a short-term follow-up for pediatric patients. Further studies with a longer follow-up period to confirm the long-term biomechanical changes of the ATE-CXL treatment are also needed.Our study suggests that ATE-CXL is a safe and effective treatment for pediatric patients with progressive keratoconus. The DCR parameters provided by Corvis ST can be used to evaluate changes of corneal biomechanics after cross-linking, which indicated that the corneal stiffness of pediatric keratoconus increased at 1 year after ATE-CXL, but the long-term effects need further observation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Eye and ENT Hospital of Fudan University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.WJ, MT, and XZha were responsible for the initial plan, study design, data collection, data extraction, data interpretation, manuscript drafting, statistical analysis, and conducting the study. LS, YS, and ML were responsible for data collection, extraction, and critical revisions of the manuscript. WJ, MT, and XZho were responsible for data interpretation, manuscript drafting, supervision, and critical revisions of the manuscript for important intellectual content. XZho was the guarantor for this article and has full responsibility for this study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor is a consultant for the manufacturer Corvis ST, and declares that the assessment of the manuscript was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the visible spectral region, the imaginary part of the refractive index becomes relevant. The refractive index in the visible wavelength range (400–600 nm) was in the range 1.7–1.80.The polymerization of 3,4-dihydroxy-L-phenylalanine leads to a carboxylic acid-rich synthetic melanin-like material (poly-L-DOPA). Synthetic melanin most resembles natural eumelanin in chemical structure. However, its deposition on surfaces leading to colored surfaces by interference is not as easy to accomplish as in the case of the preparation of colored surfaces by dopamine hydrochloride polymerization. This study deals with the preparation of new colored surfaces made from poly-L-DOPA displaying vivid colors by interference. These surfaces were obtained by depositing thin films of poly-L-DOPA on a reflective silicon nitride substrate. A high ionic strength in the polymerization medium was essential to accomplish the coating. The effect of ionic strength on the resulting surfaces was studied via reflectance, Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). The refractive index was determined by ellipsometry, and was nearly constant to 1.8 when  Many colors in nature are the result of the interaction of light with structured materials . InterfeTo date, dopamine hydrochloride has a central role in the preparation of synthetic melanins. It spontaneously polymerizes in a basic aqueous medium to produce insoluble pigments with similar properties to its natural analogous form. Similarly, polydopamine is able to coat a wide variety of substrates ,8. ThereOn the other hand, colored melanin surfaces obtained from the polymerization of 3,4-dihydroxy-L-phenylalanine (DOPA) and, consequently, the characterization of the poly-L-DOPA refractive index are seldom explored. DOPA is an important free amino acid that is the initial precursor in eumelanin biosynthesis . HoweverImportantly, it has been recently reported that poly-L-DOPA coating over different surfaces can be achieved when DOPA oxidative polymerization was conducted in an aqueous medium of high ionic strength . With thThe AFM images (5 µm × 5 µm) were obtained with a Veeco Multimode scanning probe microscope equipped with a Nanoscope IV , a controller operating in tapping mode with a phosphorus doped silicon cantilever (model RTESP). Mean square roughness values were extracted from these images. The surface images were obtained by scanning electron microscopy (SEM) using a Hitachi SU-8000 instrument . Raman spectra were obtained with a Micro-Raman Spectrometer . Surfaces were excited at 785 nm. Optical characterization: both ellipsometric and reflectometric measurements were taken with a GES 5E , provided with a variable angle goniometer, Xe-lamp, monochromator and photomultiplier. The reflectance diffuse spectrum was characterized in a Jasco 616 spectrophotometer .Crystalline silicon wafers with a thickness of 520 µm and coated with a 200 nm layer of silicon nitride (LPCVD) were purchased from Addison Engineering, Inc. The characteristics of the wafer were as follows: (i) front finish: mirror polish; (ii) back finish: etch; (iii) type: p/Boron; and (iv) orientation: (100).Time effect: The silicon wafer piece of ca 1 cm × 1 cm was immersed in 30 mL of a buffered solution of L-DOPA at pH 10  (2 mg/mL). The buffer was trizma-HCl   and contained NaCl at a concentration of 0.87 M. The polymerization was left to proceed at room temperature in an open flask with controlled magnetic agitation (200 rpm) at different times: 2, 3, 4, 5, 6, 7, 8 and 9 h. After each time, the wafer was washed with deionized water and air dried. The poly-L-DOPA aggregate dispersion was dialyzed (MWCO = 6000–8000) against water for 5 days in order to remove the buffer medium and remaining monomer molecules. After dialysis, the UV-vis absorption spectrum of the poly-L-DOPA dispersion in water was characterized. NaCl concentration effect: The same procedure as described above was used. However, coating was studied at 9 h of polymerization, while the NaCl concentration in the buffer was varied. The following NaCl concentrations were studied: 0.87, 0.40 and 0.23 M.3N4 over a silicon wafer. The surface was smooth and presented a strong reflectance at 400 nm, which was responsible for the violet color that the surface displayed; see The reflective substrate consisted of a 200 nm layer of SiPoly-L-DOPA coating over polyethylene, polyvinylidene difluoride and polytetrafluoroethylene substrates by the polymerization of DOPA in an aqueous buffer solution of low ionic strength has previously been reported . However3N4 surface. The polymerization of L-DOPA (2 mg/mL) was carried out in an aqueous buffer solution  that contained NaCl to increase the ionic strength. The deposition of poly(L-DOPA) on the silicon nitride wafer was carried out by submerging the silicon nitride wafer in an opened air-flask where the polymerization was taking place. When the concentration of NaCl was 0.23 M, the color of the surface changed to blue, and it progressively red shifted when the salt concentration was increased up to 0.87 M; see Therefore, ionic strength seemed to be a determinant factor to obtain poly-L-DOPA coatings. First, the effect of NaCl concentration (in the polymerization medium) on both the morphology and color of poly-L-DOPA surfaces was evaluated. For this purpose, different NaCl concentrations in the polymerization medium were studied: 0.23, 0.40 and 0.87 M, while the polymerization time was chosen as 9 h in order to obtain a significant amount of deposit on the SiThe experimental perpendicular component of the reflectance at 50° is shown in −1, which have previously been reported in natural melanin samples [The presence of a poly-L-DOPA deposit was confirmed by Raman spectroscopy. The Raman spectrum shows peaks at 1386 and 1530 cm samples ; Figure qR) value was 3.4 nm and the thickness of the poly-L-DOPA coating was determined to be 8 ± 2 nm. Increasing the NaCl concentration to 0.57 M led to a rougher surface with a Rq of 27 nm and a thickness of 51 ± 27 nm. The surface obtained with the highest NaCl concentration presented a greater roughness value of 63 nm. It is important to highlight that the roughness value was always lower than the film thickness.A more in-depth analysis of the salt effect on the poly-L-DOPA coating characterization of surfaces was conducted by AFM. The poly-L-DOPA film thickness was calculated by making a needle-scratch on the surface and determining the depth of the scratch (thickness of film). sR), which agreed with the characterization of the roughness effect in polydopamine films [The roughness had a strong effect on the reflectance of the surfaces. As can be seen in ne films .Although it has been stated that an increase in the NaCl concentration decreases the quality of the coating surface, the extremely low growth rate forced us to choose a compromise between quality and simplicity of production. In this regard, we fixed the NaCl concentration at 0.87 M, and the polymerization time was varied from 2 to 8 h. The image of silicon nitride wafers upon modification with poly-L-DOPA at different times is shown in SEM and AFM were used to characterize the morphology of these surfaces. SEM images of the surface as a function of polymerization time are shown in The effect of polymerization time on surface morphology could be observed more clearly in the AFM characterization. qR values are shown in qR value increased with the polymerization time, from 20 nm for 2 h, up to 60 nm for 8 h of polymerization. Additionally, surface thicknesses of 28 and 52 nm were obtained after 2 and 3 h of polymerization, respectively. After 8 h of polymerization, the coating reached 145 nm thickness. Nevertheless, the roughness values were lower than the thicknesses in all polymerization times, unlike polydopamine coatings. This might indicate that, in the case of polydopamine film, growth is caused by the deposition of nanoparticles, while in the case of poly-L-DOPA, the deposition of oligomers might be the predominant mechanism.The depth histograms are shown in qR values in the 20–60 nm range. However, to achieve similarly colored surfaces by polydopamine coating, the thickness was reported to be in the 30–170 nm range, with the qR values nearly identical to the thickness values [To summarize, the poly-L-DOPA film deposited on silicon nitride surfaces by the oxidative polymerization of DOPA in the presence of NaCl 0.87 M led to colored surfaces from blue to magenta when the film thickness was in the range 28–145 nm and s values .The determination of the refractive index of melanins has been a matter of study in the last two decades . It has ρ = pR/sR were fitted simultaneously in a spectral range from 250 to 850 nm; see n > 1.8 for λ > 650 nm). Right at the red spectral region (λ < 640 nm), the imaginary part of the refractive index became relevant, increasing its value for blue spectral regions. The refractive index decreased in the visible wavelength range (400–600 nm) gradually from 1.8 to 1.7. These values were slightly lower than the reported ones [We used colored surfaces to determine the reliability of the characterized refractive index of poly-L-DOPA over a silicon substrate, since all optical properties, including color, are determined by the refractive index of the deposit. When the substance appears as a homogeneous thin layer, spectroscopic ellipsometry is the best tool. In order to achieve a greater accuracy, a thin and homogenous layer of poly-L-DOPA was grown on a silicon wafer, and eight spectra were taken at angles from 45 to 80° each 5°, so that we had data below and above the Brewster angle of silicon. The eight complex spectra of ted ones .Knowledge of the refractive index allowed us to predict the optical response in different shapes as aqueous suspensions or thin film deposits. More specifically, we could reproduce the optical reflectance spectra by using the fitted data in a transfer matrix calculation  Supplem. This prThe chemical composition and structure of both naturally occurring and synthetic melanins remains elusive ,24. This−4. The experimental spectrum was simulated taking into account the Mie theory [α = 4πk/λ [The aggregates of poly-L-DOPA in water presented a broad declining absorbance with the wavelength in the 200–800 nm range without any spectral characteristic in agreement with prior works dealing with synthetic melanins , but a be theory ,27 by coe theory ,29; DHICe theory . The lesNew colored poly-L DOPA surfaces were prepared. Poly-L-DOPA was deposited on a reflective silicon nitride substrate by immersing the wafer in the DOPA polymerization medium (pH = 8.5) that had a high ionic strength. The use of a reflective substrate permitted us to obtain visual information about the coating process and thickness uniformity of the deposited poly-L-DOPA film. An ionic strength was necessary to achieve the substrate coating. When the concentration of NaCl was 0.23 M, a high-quality surface was obtained with a roughness of 6 nm. An increase in ionic strength led to an increase in film thickness and surface roughness.qR values were in the 20–60 nm range. The roughness values were lower than the thicknesses at all polymerization times, which may indicate that during film growth, the poly-L-DOPA deposition of oligomers may be the predominant mechanism.For a NaCl concentration of 0.87 M, the control of the polymerization time permitted us to control the film growth; colors changed from blue to magenta when the film thickness was increased from 28 to 104 nm. The n > 1.8 for λ > 650 nm). The imaginary part of the refractive index become relevant at the red spectral region (λ < 640 nm). The refractive index in the visible wavelength range (400–600 nm) gradually ranged from 1.7 to 1.8.These surfaces, which were uniform in color and concomitant in thickness, were used to determine the refractive index of poly-L-DOPA by ellipsometry spectroscopy. Poly-L-DOPA presented a very high and nearly constant refractive index at IR wavelengths (The refractive index obtained in this work permitted the reproduction of the reflectance spectra of surfaces, the absorbance spectra for poly-L-DOPA film, and poly-L-aggregates dispersed in solution.The newly colored poly-L DOPA surfaces have great potential for the development of future photonic materials. Poly-L-DOPA presents a higher chemical versatility than polydopamine. In addition to the polydopamine chemistry, a poly-L-DOPA coating is rich in carboxylic acids, and new materials may be obtained from these new surfaces.


The median time to seroconversion of NAbs was 5.5 days post onset of symptoms. The rate of positive sVNT was 52% in the first week, reached 100% in the third week, and remained above 97% till 6 months post onset. Quantitatively, NAbs peaked in the fourth week and only a quarter of patients had an estimated peak titer of >1000. NAbs declined with a half-time of 61 days (95% CI: 49–80 days) within the first two months, and the decay deaccelerated to a half-time of 104 days (95% CI: 86–130 days) afterward. The peak levels of NAbs were positively associated with severity of COVID-19 and age, while negatively associated with serum albumin levels. The observation that the low-moderate peak neutralizing activity and fast decay of NAbs in most naturally infected individuals called for caution in evaluating the feasibility of antibody-based therapy and vaccine durability. NAbs response positively correlated with disease severity, warning for the possibility of repeat infection in patients with mild COVID-19.Our understanding of the protective immunity, particularly the long-term dynamics of neutralizing antibody (NAbs) response to SARS-CoV-2, is currently limited. We enrolled a cohort of 545 COVID-19 patients from Hubei, China, who were followed up up to 7 months, and determined the dynamics of NAbs to SARS-CoV-2 by using a surrogate virus neutralization test (sVNT). In our validation study, sVNT IC Thus, understanding neutralizing responses, especially NAbs to RBD of SARS-CoV-2, is essential in determining the onset of humoral immunity, evaluating potential capacity of viral clearance, identifying donors for convalescent plasma therapy, and assessing vaccine efficacy during clinical trials or after large-scale vaccination.The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2).However, our understanding of the protective immunity to SARS-CoV-2 is currently limited. Little is known about the dynamics of RBD-targeting NAbs, including its generation, longevity, and correlation with the clinical settings, particularly at population level.Lack of reliable and versatile tests for assessment of neutralizing activity remains a big challenge. The conventional virus neutralization tests, such as focus reduction neutralization test (FRNT) and pseudo-virus-based neutralization test remain the only platforms for detection of NAbs. Both tests require live viruses and cells, and days to obtain results, and thus unsuitable for large-scale measurement and population study.6 sVNT quantifies RBD-targeting NAbs without the need for biosafety level 3 containment or any live virus or cells. Here, by using this sVNT, we assessed the dynamics of RBD-targeting NAbs response to SARS-CoV-2 in a cohort of 545 COVID-19 patients up to 7 months post onset of symptoms, and explored the clinical correlates to the peak NAbs response in individuals.Recently, a surrogate virus neutralization test (sVNT) has been established based on antibody-mediated blockage of ACE2-spike protein-protein interaction.th, 50th, and 75th percentiles of follow-up time were 45 days, 62 days, and 175 days, respectively, post onset. The flowchart of sample selection was presented in Supplementary Fig. Our study cohort included 545 COVID-19 patients who were diagnosed between January and March 2020 and managed at the designated hospitals in Honghu and Jingzhou, Hubei province. All cases were confirmed by RT-PCR testing. Of 545 patients, the mean age was 50.9 years, 286 (52.5%) were males, 54 (9.9%) were severe, and 379 (69.5%) had at least two time points of sera collection during the course of study. The 2550) serve as a surrogate for the titer (IC50) obtained from FRNT? and (3) could measurement from sVNT at a single dilution (1:20) or total binging IgG level serve as a surrogate for FRNT titer?We performed validation study on sVNT to address the following three questions: (1) what is the specificity and sensitivity of this assay measured in 1:20 diluted sera? (2) could sVNT titer (IC20) using sera from 194 healthy individuals as negative controls, and sera from 395 COVID-19 patients collected on days 21–60 post onset of symptoms as positive controls, resulting in a specificity of 100%  and a sensitivity of 97.2%  when using a cutoff value of 20% for NR20.First, we evaluated the specificity and sensitivity of sVNT when measuring neutralization rate in 1:20 diluted sera ], had a log-linear relationship with both FRNT and sVNT titers, with correlation coefficients of 0.84 and 0.95, respectively  = 4.5 + 0.88 × logit (NR20) (Equantion 1), where log denotes the natural logarithm. Logit NR20 values of 0.12, 1.38, and 2.74 were equivalent to a FRNT titer of 100, 300, and 1000, respectively. As a result, we chose to use logit NR20 as a surrogate for log titers of NAbs in subsequent studies.We then evaluated the relationship of antibody  levels measured at 1:20 sample dilution with FRNT titers. The logit transform of NRly Figs.  and 1C. ly Figs. . The fac20 of 2.2 , and slowly declined afterward  of peak logit NR20 was 2.2 (1.3 to 2.8), which was equivalent to a FRNT titer of 624 , respectively, indicating that low-moderate levels of NAbs were observed in 75% of patients in this cohort.We calculated the peak titer of RBD-targeting NAbs in a given individual as the highest titer observed between 2120 > 1.3, the 25th percentile) and at least one additional antibody measurement after the peak  in 197 COVID-19 patients who participated in the 6-month visit. The follow-up time ranged from 144 to 206 days post onset, with a median of 180 days. The distribution of the titers was presented in Supplementary Fig. We measured FRNT titers (dilution at IC20). A total of 57 demographic and clinical features were analyzed, including age, gender, disease severity and duration of COVID-19 (days from onset to the earliest-time RT-PCR testing turning from positive to negative), presence of symptoms, imaging signs of lung infection, comorbidities, complete blood count, and laboratory biochemistry tests  and is summarized in Table 20 compared with that without imaging signs. Age and duration of COVID-19 were also positively associated with peak NAbs, while serum albumin level was negatively associated with peak NAbs .In this study, we described for the first time the 6-month dynamics of the NAbs response to SARS-CoV-2 in a cohort of COVID-19 patients. Consistent with the previous reports, seroconversion is generally completed within 2 weeks.et al reported a faster decay of SARS-CoV-2-IgG with a half-time of 36 days over 3 months in 31 patients with mild COVID-19.10 Several reasons may explain this discrepancy. First, the study populations differ. Ibarrondo’s study only included mild patients, while our study enrolled patients with various clinical settings. For example, our cohort was on average 7 years older. We have shown that younger age is associated with faster decay of antibodies. Second, we used linear mixed-effect model with random slope and random intercept to estimate the slope, while Ibarrondo’s study used simple linear regression. Third, existence of ethnic difference in the decline rate could not be excluded. The trend in RBD-targeting NAbs dynamics was similar to that of IgG antibody observed in the same cohort and in the previous report.9 NAbs targeting RBD are likely the crucial component of the protective immunity against SARS-CoV-2.12 The observed fast decay of NAbs in the most naturally infected individuals calls for caution in evaluating the feasibility of antibody-based therapy for COVID-19 and vaccine durability.Studies on the long-term evolution of antibodies to SARS-CoV-2 remain limited. It is still unclear how long the virus-specific antibodies may persist after infection with SARS-CoV-2. In our study cohort, at 6 months post onset, 97% were still tested positive for NAbs, though on average, the level of NAbs was dropped by one natural log unit  from the peak. In the first two months post onset, NAbs declined with a half-time of 61 days. After 2 months, it deaccelerated to a half-time of 104 days. A recent study in 30 COVID-19 patients found that NAbs titers declined by 34.8% within 3 months post onset, which was consistent with our observation. Decline of IgG appeared to be time-independent, with a decay half-time of 95 days (95% CI: 85–108 days). Recently, Ibarrondo 13 These results sound a warning for the possibility of repeat infection in patients with mild COVID-19.The availability of detailed medical information of the study cohort enabled us to identify clinical settings that are mostly associated with the peak NAbs. Unexpectedly, we found that disease severity, indicated by obvious symptoms, pulmonary lesions, and longer virus shedding time, was positively associated with peak NAbs response. In addition, age was positively correlated with the peak-neutralizing activity, while the level of serum albumin was negatively correlated. These may also be explained by the association between disease severity and the NAbs response since both patients with old age and low albumin are more susceptible to severe disease. Our findings were consistent with a recent report that severity was positivity correlated with NAbs response.A strength of our study was the large sample size, including patients with various clinical settings and longitudinal tracking of NAbs up to 7 months, making it feasible to accurately estimate the dynamics of NAbs response and compare the responses among clinic subgroups. In addition, few new COVID-19 cases were reported in Hubei province since May, making it very unlikely that our analysis of long-term NAbs response was confounded by re-infection. This study also has limitations. The longest follow-up time was 7 months. Longer follow-up study is required to assess the long-term dynamics of NAbs after this period. Our cohort was exclusively Chinese. Whether the findings from our study are generalizable to other populations requires further validation.In summary, we have determined the dynamics of RBD-targeting NAbs response to SARS-CoV-2 up to 7 months post onset in a large cohort of COVID-19 patients. Most patients have low-moderate peak-neutralizing activity. NAbs declines quickly in the first two months post onset, and the decline deaccelerates afterward with a half-time of 104 days. NAbs response positively correlated with disease severity. Our data have thus provided important insights for humoral protective immunity against SARS-CoV-2.n = 545) who had been diagnosed between January and March and managed at five local hospitals designated for the care and management of COVID-19 patients in Honghu and Jingzhou, Hubei province. All cases were confirmed by RT-PCR testing. According to the Health Commission of Hubei province, until end of March, a total of 1580 COVID-19 cases had been reported from Honghu and Jingzhou of Hubei province. So, our cohort represented 34% of the COVID-19 cases in the region. Demographic and clinical information, including gender, age, history of diseases, and presence of symptoms at onset, were extracted from the electronic medical records. Reports of chest imaging examination and laboratory tests, including SASR-CoV-2 RT-PCR testing, were exported from the hospital imaging and laboratory information systems. Under the regulation for COVID-19 management, these patients were isolated and managed at the designated hospitals until viral shedding. After discharge, patients were followed up biweekly until 60 days post onset. In late July, we conducted another round of follow-up in which 197 convalescent patients participated. Serum samples were collected in local hospitals. All samples were inactivated at 56 °C for 30 min and stored at −80 °C before use. In addition, we have collected sera from 194 healthy individuals who took regular physical checkup at Nanfang hospital in May and were tested negative for both RT-PCT testing and total binding IgG and IgM assay. Sera from these individuals were used as negative controls in the validation study. The flowchart of sample selection was presented in Supplementary Fig. The study cohort consisted of all COVID-19 patients  following the manufacturer’s instruction. Briefly, diluted serum samples (or negative and positive controls) were 1:1 mixed with the diluted HRP-RBD (the horseradish peroxidase-conjugated recombinant SARS-CoV-2 RBD fragment) solution, and incubated at 37 °C for 30 min. Add 100 μl each of the mixture to a well in a 96-well plate precoated with human ACE2 receptor protein, and incubate at 37 °C for 15 min. Wash the plate with 260 μl of 1× Wash Solution for four times, add 100 μl of TMB Solution to each well, and incubate the plate in the dark at 25 °C for 15 min. Add 50 μl of Stop Solution to each well to quench the reaction, read the absorbance at 450 nm in a microtiter plate reader immediately. The neutralization rate (NR) was calculated as 50 of surrogate virus neutralization was calculated from NR values of serially diluted samples using the 4-parameter logistic model.14While FRNT remains the gold standard for serological testing and determining immune protection, it is not practical for large scale epidemiological studies due to its low throughput and stringent biosafety requirement. sVNT is an assay detecting NAbs against SARS-CoV-2 that blocks the interaction between the RBD of the viral spike protein with the ACE2 cell surface receptor.13 Fifty-μl serum samples were serially (1:4) diluted, mixed with 50 μl of SARS-CoV-2  in 96-well microwell plates, and incubated for 1 h at 37 °C. Mixtures were then transferred to 96-well plates seeded with Vero E6 cells  for 1 h at 37 °C. Inoculums were then removed before adding the overlay media . The plates were then incubated at 37 °C for 24 h. Overlays were removed and cells were fixed with 4% paraformaldehyde solution for 30 min. Cells were permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG  for 1 h at room temperature before adding HRP-conjugated goat anti-rabbit IgG(H + L) antibody (1:4000 dilution) . Cells were further incubated at room temperature. The reactions were developed with KPL TrueBlue Peroxidase substrates . The numbers of SARS-CoV-2 foci were calculated using an EliSpot reader . IC50 of virus neutralization was calculated using the 4-parameter logistic model.SARS-CoV-2 FRNT was performed in a certified biosafety level 3 lab as previously described.The total binding IgG and IgM antibodies against SARS-CoV-2 were measured using a commercially available Magnetic Chemiluminescence Enzyme Immunoassay kit  according to the manufacturer’s instructions. Antibody levels were expressed as the ratio of the chemiluminescence signal over the cutoff value (S/CO). A S/CO value of >1.0 for either IgG or IgM was regarded as positive.th, 75th percentile), and categorical variables as counts and percentages.Continuous variables were presented as mean±sd or median , gender, and peak level (by median of the peak) by adding interaction terms in the model. Decay half-time was calculated as log(0.5)/slope.We defined the peak of the antibodies as the highest level observed between 21NR20), we used a stepwise linear regression model with Bayesian Information Criteria (BIC) to select variables from a pool of 57 demographic and clinical features, including age, gender, severity and duration of COVID-19 (days from onset to the earliest-time RT-PCR testing turning from positive to negative), presence of symptoms, imaging signs of lung infection, comorbidities such as hypertension, diabetes, and coronary heart disease, complete blood count, and common blood laboratory chemistry tests such as glucose, lipids, electrolytes, liver and kidney functions, and albumin and globulin. A complete list of the variables could be found in Supplementary List To identify clinical correlates that were associated with the peak titer of NAbs Supplemental material


LncRNA dysregulation and the tumor microenvironment (TME) have been shown to play a vital role in the progression and prognosis of colon cancer (CC). We aim to reveal the potential molecular mechanism from the perspective of lncRNA in the TME and provide the candidate biomarkers for CC prognosis.ESTIMATE analysis was used to divide the CC patients into high and low immune or stromal score groups. The expression array of lncRNA was re-annotated by Seqmap. Microenvironment-associated lncRNAs were filtered through differential analysis. The m6A-associated lncRNAs were screened by Pearson correlation analysis. Lasso Cox regression analyses were performed to construct the m6A- and tumor microenvironment-related lncRNA prognostic model (m6A-TME-LM). Survival analysis was used to assess the prognostic efficacy of candidate lncRNAs. Enrichment analyses annotated the candidate genes’ functions.We obtained 25 common differentially expressed lncRNAs (DELs) associated with immune microenvironment and m6A-related genes for subsequent lasso analysis. Four out of these DELs were selected for the m6A-TME-LM. All the four lncRNAs were related to overall survival, and a test set testified the result. Further stratification analysis of the m6A-TME-LM retained its ability to predict OS for male and chemotherapy adjuvant patients and performed an excellent prognostic efficacy in the TNM stage III and IV subgroups. Network analysis also found the four lncRNAs mediated co-expression network was associated with tumor development.We constructed the m6A-TME-LM, which could provide a better prognostic prediction of CC. Colon cancer (CC) is a refractory malignant tumor. The incidence of CC is increasing at an alarming rate due to the rapid industrialization of recent times and the changes in urban lifestyles worldwide. Almost 1.5 million people were diagnosed with colon cancer each year, and more than 500,000 deaths occurred every year. CC patients also accounted for 40% of cancer cases diagnosed each year . ChemothAs a new treatment strategy, the treatment of tumor microenvironment (TME) has attracted public attention . TME is ALKBH5 was downregulated in human colon cancer tissues and was significantly correlated with distant metastasis. A functional experiment found that overexpression of ALKBH5 inhibited colon cancer cells invasion in vitro and metastasis in vivo (PD-L1 mRNA and the binding of FTO to the PD-L1 mRNA was testified in HCT-116 cell line by RNA immunoprecipitation assay which indicated that FTO could regulate PD-L1 expression in colon cancer cells (OCC-1 suppresses cell growth through destabilizing HuR (ELAVL1) protein in colorectal cancer  modification, an epigenetic methylation modification that plays a crucial role in RNA transport, translation, and other functions , 12. M6A in vivo ; the m6Aer cells ; and lncferation .SOX9-activated FARSA-AS1 is reported to affect cell growth, stemness, and metastasis in colorectal cancer through upregulating FARSA and SOX9 (LOC441461 (STX17-AS1) could modulate colorectal cancer cell growth and motility . Then, the lncRNA expression matrix of the TCGA CC data set was used as an independent test data set and downloaded from the TANRIC database . Moreover, the human genome annotation file (GRCh38/hg38) was downloaded from the UCSC database (http://hgdownload.cse.ucsc.edu/).Annotation files for lncRNAs and protein-coding genes (PCGs) were downloaded from GENECODE . First, the sequence of 54675 probes (HG-U133_Plus_2) was mapped to GRCh38 human reference genome by Seqmap, and the corresponding genome position of these probes was found. Then, remove probes that match to multiple locations, leaving probes that uniquely mapped to a single location in human genome with a maximum of two mismatches. Finally, according to the downloaded lncRNA genome location information, the probe of the previous step was corresponding to the lncRNA. A total of 4674 probes from the HG-U133_Plus_2 array were uniquely mapped to lncRNAs. In the end, we constructed the lncRNA expression profile include 3724 lncRNAs in these data sets.We mapped the probe sequences to the human genome (hg38) by Seqmap software  were identified with an adjusted P-value < 0.05 and an absolute log2 fold change ≥ 0.263.On the other hand, we also divided the patients into high- and low-risk score groups based on our m6A-TME-LM and analyzed the differentially expressed genes in the groups. The differentially expressed genes (DEGs) were identified with an adjusted P-value < 0.05 and an absolute log2 fold change ≥ 0.585.The expression matrix for 21 m6A-related genes was extracted from two separate data sets based on previously published articles. Then, Pearson correlation coefficient (PCC) was used to evaluate the expression correlation between these m6A-related genes and DELs. LncRNAs with PCC ≥ 0.3 or PCC ≤ −0.3 and P-value < 0.05 were selected as m6A-related lncRNAs. Only lncRNAs that meet the threshold in the two data sets will be selected for subsequent analysis.The m6A-associated differential lncRNAs filtered by two data sets were intersected to get 25 common m6A-associated lncRNAs. Subsequently, univariate cox regression analysis was used to screen out the prognostic lncRNAs. Next, LASSO Cox regression was performed using the R package “glmnet”  to constKaplan-Meier analysis was used to screen vital prognostic lncRNAs. Survival curves were drawn to illustrate the associations of expression levels of these lncRNAs with CC outcome by the “survival” R package. The multivariable Cox regression analysis was used to test whether the signature was independent of other clinical features. Using pROC package, the area under the ROC curve (AUC) was calculated to assess the prognostic efficiency of this signature.The survival analysis was also used to compare OS between different subgroups, including low-risk and high-risk subgroups, based on the expression of four m6A-associated lncRNAs. The subgroups were separated by the following features: chemotherapy adjuvant (yes or no), gender , TNM stage (I and II or III and IV), and age (≤ 68 or > 68 years).Based on the m6A-TME-LM risk score, we separated the CC patients into low- and high-risk groups. Next, differentially expressed genes (DEGs) associated with the four lncRNAs in m6A-TME-LM were screened from mRNA and lncRNA expression profile data using Pearson correlation coefficient (PCC). Those dysregulated lncRNA-mRNA pairs with PCC ≥ 0.3 or PCC ≤ −0.3 and p < 0.05 were selected as co-expressed lncRNA-mRNA pairs and used to construct the co-expression network.All the DEGs and the DELs related genes in the study were extracted for further functional enrichment by using the “clusterProfile” R package  and Meta. Similarly, in GSE17536 data set, there were 25 immune-associated DELs and 23 stromal associated DELs . Then, we obtained the expression levels of 21 m6A-associated genes through the two data sets separately and performed Pearson correlation analysis between these genes and our m6A-associated lncRNAs in each data set. The lncRNAs with expression values associated with the 21 m6A-associated genes (p < 0.05 and |PCC| > 0.3) were defined as m6A-associated lncRNAs. Finally, we obtained 78 lncRNAs that were significantly associated with m6A in GSE39582 and 36 eligible ones in GSE17536. Finally, 25 m6A-associated lncRNAs were selected in both two data sets .A total of 3724 lncRNAs’ expression levels were re-annotated in two CC data sets. After the ESTIMATE analysis, we divided the CC patients into high- and low-score groups based on the immune or stromal score. Then, we used differential expression analysis to get DELs associated with tumor microenvironments. In GSE39582 data set, there were 86 immune-associated DELs, and 78 lncRNAs were differentially expressed according to immune or stromal score . Similar. Similar. Similar.Combined with clinical information, we screened prognosis-related lncRNAs from the 25 candidate lncRNAs by univariate Cox regression analysis (p < 0.05). We obtained 12 out of the 25 lncRNAs were significantly associated with overall survival (OS) of CC patients . The risk score of each sample in the data set was calculated based on the coefficient of four lncRNAs involved in m6A-TME-LM . Then, patients were divided into two subgroups based on the median risk score (low-risk and high-risk). The survival curve showed that patients with higher risk scores had a worse prognosis . And the ROC curves likewise found that m6A-TME-LM had a good efficacy to predict survival time in this data set. .We then performed LASSO Cox analysis based on the 12 prognostic lncRNAs in the GSE38592 data set and generated a model which contains four lncRNAs . The ris. The ris. The ris. The ROC curve also indicated that this model had a more vital prognostic ability in the data set . Meanwhile, an independent test data (TCGA data set) was used to testify the predictive efficacy of the model further. We also got consistent results . The ROC curve also indicated that this model had a stronger prognostic ability in the data set (AUC = 0.986). These results demonstrated the m6A-TME-LM had a stable predictive power of prognosis in CC.To testify the prognostic efficacy of m6A-TME-LM, we conducted the model in GSE17536 data set using the same algorithm. The low-risk and high-risk subgroups were also distinguished based on the median risk scores of CC patients in this data set. Ultimately, we obtained results consistent with the training data set . The ROC. The ROC. The alluvial plot showed the roles of these four lncRNAs in CC . Then, survival curves showed that all the four lncRNAs are survival-associated in both two data sets .Four crucial lncRNAs in the model were evaluated by univariate Cox regression analysis. The forest plot shows that ENSG00000254290 is a protective factor with HR (Hazard ratio) < 1, while ENSG00000237187, ENSG00000234456, and ENSG00000233901 are risk factors with HR> 1 in CC patients . The all. The all. Similarly, we confirmed that m6A-TME-LM retained its prognostic efficacy of male patients  and patients with chemotherapy adjuvant . Patients with higher risk also had a worse OS in both the TNM stage III and IV subgroups . These data proved that the model could be a novel predictor and performed an excellent prognostic efficacy in CC. The m6A-TME-LM may help to improve the prognosis of CC patients.To further assess the prognostic efficacy of m6A-TME-LM, a stratified analysis was used to explore whether the model still could predict OS in different subgroups. The result showed that the higher risk CC patients had a worse survival rate in age > 68 subgroups . Similar. Similar. Similar. Heatmap showed the cluster efficiency of these DEGs . Then, enrichment analysis was performed to confirm the function of the m6A-TME-LM. As shown in . Further enrichment analysis in Hallmarks showed that these genes involved in the inflammatory response, KRAS signaling and interferon Gamma response, etc. . All of these suggest that the m6A-TME-LM plays an essential role in CC.To better confirm the signature’s potential functions in CC, we classified the samples into high and low risk‐score groups based on the m6A-TME-LM and obtained DEGs influenced by the model . Heatmap. Heatmap. Heatmap. Heatmap. Next, the mRNAs in the network were upload to the ClueGO tool of Cytoscape for functional analysis, and the result showed that these genes were enriched in focal adhesion, AGE-RAGE signaling pathway, Toll-like receptor signaling pathway, and NF-kappa B signaling pathway, etc. .To further explore how these crucial lncRNAs regulate the aberrant expression of mRNAs in CC, we constructed a co-expressed network based on the correlation between the TME-m6A-related lncRNAs and DEGs. Ultimately, four lncRNAs and 906 mRNAs were selected in the network . Next, tMETTL3-mediated m6A methylation modification directly promotes YAP translation and increases YAP activity by regulating the MALAT1-miR-1914-3p-YAP axis to induce drug resistance and metastasis in NSCLC  was identified as the potential lncRNA biomarkers for liver hepatocellular carcinoma (LIHC) (NR2F1-AS1) promoted the progression of NB through the miR-493/TRIM2 axis. It also regulates miR-371a-3p/TOB1 axis to suppress the proliferation of CRC cells (RP11-65J3.1) is a novel lncRNA that only puts forward in this study, and the potential function needs further analysis. ENSG00000234456 (MAGI2-AS3) has been found to be involved in multiple cancers, including esophageal cancer  . The preRC cells . ENSG000l cancer , breast l cancer , and coll cancer , 39, theFirst, we identified the immune-related DELs by ESTIMATE analysis and obtained several m6A-related lncRNAs by correlation analysis. Then, we developed the m6A-TME-LM by lasso regression. Next, survival analysis was used to find potential prognostic biomarkers, and four lncRNAs with independent prognostic efficacy were obtained. Finally, the co-expression network of these lncRNAs helps us to find the potential functions of the crucial lncRNAs. This study may be helpful for future studies concerning CC with the aim of finding potential prognostic targets of it.The original contributions presented in the study are included in the article/HZ, LZ, TL, and XD conceived and devised the study. HZ, LZ, and SL performed bioinformatic and statistical analysis. JW and CF found testify data and analysis tools. HZ, TL, and XD supervised research and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (Research Index: 81871317), “Winter Olympic Emergency Medical Support” (Research Index: 2019YFF0302300) sponsored by Ministry of National Science and Technique, and Project (RDE2020-15) supported by Peking University People’s Hospital Scientific Research Development Funds.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MsZF-HD genes in the alkaline stress response, this paper assessed biological information and performed transcriptome analysis of the MsZF-HD gene family by using the genomes of two different varieties of alfalfa (XinJiangDa Ye and Zhongmu No. 1).Zinc finger homeodomain (ZHD) protein is a plant-specific transcription factor and a potential regulator of phosphoenolpyruvate carboxylase (PEPCase)-coding genes, and it also participates in plant growth regulation and abiotic stress responses. To study the function of MsZF-HD genes were identified in the two different varieties respectively, including the alleles of XinJiangDa Ye. According to their phylogenetic relationships, the 60 MsZF-HD genes were divided into 5 ZHD subfamilies and 1 MIF subfamily. A total of 88.3% of MsZF-HD genes do not contain introns and are unevenly distributed among the 6 chromosomes of alfalfa. A collinearity analysis indicated that 26 genes of XinJiangDa Ye have no orthologous genes in Zhongmu No. 1, although these genes  have homologous genes in Arabidopsis thaliana, Medicago truncatula and Glycine max. Through RNA-seq and qRT–PCR verification, it was found that MsZF-HD genes are downregulated to participate in the alkaline stress response.In total, 49 and 11 MsZF-HD genes and provide a theoretical basis for revealing the difference between XinJiangDa Ye and Zhongmu No. 1 at the genome level.The results of this study may lay the foundation for the cloning and functional study of The online version contains supplementary material available at 10.1186/s12864-022-08309-x. ZF-Hng genes . The aming genes , 4. In ang genes . Interesng genes . Unlike ng genes . Thus, tvinifera , Glycinecine max , Solanumpersicum , Fagopyrataricum , Triticuaestivum , etc.4 species [A. thaliana, 17 ZF-HD family genes have been shown to have great significance in the development of flowers [AtZHD1 is induced by drought, salinity and abscisic acid (ABA), and the overexpression of ZF-HD1 along with NAC genes confers tolerance to drought stress. It was also found that ZF-HD1 binds to the promoter of EARLY RESPONSE TO DEHYDRATION STRESS 1 (ERD1) [AtMIF1 is overexpressed in Arabidopsis, suggesting that AtMIF1 may be involved in the regulation of plant development by multiple hormones. It is possible that AtMIF1 interacts with ZHD proteins via the ZF domain and its overexpression interferes with the normal functions of ZHD proteins [GmCaM4) and are highly expressed under pathogen inoculation [OsDREB1B (DROUGHT RESPONSE ELEMENT BINDING 1B) and show differential transcriptional levels with OsDREB1B under different abiotic stress conditions, which is illustrative of the crosstalk between stress signaling pathways [OsZHD1, 2, 4, and 8 could form homo and heterodimers, and dimerization may play a prominent transactivation role in the regulation of the OsDREB1B response to abiotic stress.ZF-HD proteins are expressed predominantly or exclusively in floral tissue, indicating a likely regulatory role during floral development . The ZF- species  and in p species . Additio species . In A. t flowers . AtZHD1 1 (ERD1) . Anotherproteins . Two soyculation . In ricepathways . AdditioMedicago sativa L.) is the major forage protein source for livestock and has been planted in more than 80 countries around the world, with a planting area of more than 30 million hectares [ZF-HD family has not been comprehensively analyzed in alfalfa. Hence, we used the genome of two different varieties of alfalfa to conduct a genome-wide scan and bioinformatics analysis of the MsZF-HD genes. The analysis of this article may provide a valuable clue for further revealing the biological functions of ZF-HD family genes and the cloning of MsZF-HD genes.Alfalfa . For the 60 putative MsZF-HD proteins, their molecular weight (Mw) and isoelectric point (pI) values were also determined by the Expasy online service  was found on chromosome 8.1 in XinJiangDa Ye and abundant genes were involved in fragment repeat events  and M. truncatula (15), and 7 MsZF-HD genes of Zhongmu No. 1 were collinear with M. truncatula and G. max genes, followed by A. thaliana (5). The collinear relationship between the MsZF-HD genes of Chr5 and that of the other three plants was significantly greater compared with that of the other chromosomes.To further infer the phylogenetic relationship between alfalfa and closely related dicotyledonous plants, we analyzed the collinear relationships between alfalfa and the other three plants Fig. . The resMsZF-HD gene was analyzed to determine their potential regulatory mechanisms by using online Plant CARE software. Several light response, hormone response and stress response elements were relatively highly abundant among these cis-elements  and ABA (ABRE) response elements, which are endogenous growth regulators of higher plants and have physiological effects, such as inhibiting plant growth and germination, promoting senescence, and improving resistance . These elements include 5 ABREs in MsZHD-X1–7 and 6 CGTCA motifs in MsZHD-X1–3, MsZHD-X1–5, and MsZHD-X2–13. Stress response elements can be divided into five categories: MBS, ARE, LTR, TC-rich repeats and WUN-motif. Among them, AREs (anaerobic response elements) were the most abundant, MsZHD-X4–5 and AtZHD9 all contained 6 AREs, and almost 80% of the upstream MsZF-HD genes contained ARE elements. In addition, Box-4, GT1 motif, and G-box that respond to light were also prominent in the upstream of MsZF-HD genes, especially Box-4. 90baifenzhi of the upstream of MsZF-HD genes contained BOX-4 element, and the number of these motifs was also the largest compared with the other motifs.The identification of cis-regulatory elements in the promoter region can provide references for tissue-specific or stress-response expression patterns of genes. The 2.0-kb promoter region located upstream of the transcriptional start site (ATG) of each nts Fig. . The cisMsZF-HD genes respond to abiotic stress, the transcriptome data of Zhongmu No. 1 under different abiotic treatments  were obtained from the NCBI and assembled into the genome of Zhongmu No.1  of three genes indicated that they were not expressed in any tissue while the remaining 8 genes were expressed at least once. Among them, most MsZF-HD genes were differentially expressed in all the examined treatments. For example, MsZHD-Z5 showed a tendency of upregulation under all three treatments, while most genes  showed a low expression level. The expression of MsMIF-Z3 was upregulated under the ABA treatment, while the expression patterns under the mannitol and NaCl treatments were the opposite.To determine whether map Fig. A. Of theMsZF-HD genes under alkaline stress  belonging to group II was extremely low. ZHD II and III were almost all concentrated in group III, with low expression level. To further confirm the RNA-seq result of these MsZF-HD genes, qRT–PCR was performed for the MsZF-HD genes from different clusters , G. max (36), T. aestivum (37), F. tataricum (20), V. vinifera (13), S. lycopersicum (22), Gossypium hirsutum (35), Brassica napus (62), etc. In this study, 49 and 11 MsZF-HD genes were identified in XinJiangDa Ye and Zhongmu No. 1 by scanning the whole genome of two varieties of alfalfa, respectively. In XinjiangDa Ye, the number of MsZF-HD genes was significantly more than that of Arabidopsis and other plants. That was mainly due to the fact that the genome of XinJiangDa Ye is an allele-aware chromosome-level genome consisting of 32 allelic chromosomes. In hence, MsZF-HD genes identified include a portion of allelic genes. In our judgment, there are 10 pairs of allelic genes which can be clearly observed in Fig. MsZF-HD genes, which is significantly more than that of Zhongmu No. 1. The number of MsZF-HD genes in XinJiangDa Ye was much more than that of Zhongmu No. 1, which can lead to the formation of a flexible combinatorial system of transcription factors and may allow for subtle adjustments to many different environmental conditions because of the different binding to cis-elements and flexible dimerization [With the rapid development of high-throughput sequencing technologies, the , G. max 6, T. aesrization . In addiataricum , V. viniM. sativa and Arabidopsis, a phylogenetic tree was constructed using all ZF-HD amino acid sequences of XinJiangDa Ye, Zhongmu No. 1 and Arabidopsis. The branches on these phylogenetic tree branches were similar to those in a previous Arabidopsis report, which means that most ZF-HD proteins have homology and ZF-HD proteins are relatively conserved among different plants [M. truncatula, the number of MsZF-HD gene homologous genes in M. truncatula is not greater than that in G. max. This finding may be related to the greater number of gene duplication events in G. max, which leads to more ZF-HD genes. Some MsZF-HD genes are related to at least two pairs of homologous genes, such as MsZHD-X3–2, MsZHD-X3–5 and MsZHD-Z7, which may play an important role in the evolution of the MsZF-HD family. In addition, there are 26 MsZF-HD genes in XinJiangDa Ye that do not have orthologous genes in Zhongmu No. 1. However, those genes specific to XinJiangDa Ye  have homologous genes in the genomes of other dicotyledonous plants. It was proposed that increases or decreases in the genes on a single chromosome in the karyotype have a greater impact on the phenotype than changes in the whole genome [MsZF-HD genes. For example, Arabidopsis thaliana experienced αand βduplication in evolution which led to the expansion of gene family members [MsZF-HD genes were not observed in Zhongmu No. 1, which is consistent with the fewer MsZF-HD genes in Zhongmu No. 1, thus indicating that there were more active gene duplication events in XinJiangDa Ye.To study the developmental and evolutionary relationship between t plants . Althouge genome . Therefo members . Tandem MsZHD-X2–3, MsZHD-X3–2, MsZHD-X2–13, MsZHD-X2–14 and 6 MIF genes do not contain motif 2 or 3. A SMART online analysis showed that motif 2 and motif 3 were in the ZF-HD_dimer domain and indicated that they may be involved in the formation of homo or heterodimers and a zinc finger [MsZF-HD genes have no introns, revealing that they play an important role in the growth and development of alfalfa. This result is consistent with a previously reported result showing that the ZF-HD genes have almost no introns [MsZF-HD genes may be obtained by mutations during inheritance or evolution. The number of genes containing introns in XinJiangDa Ye is greater than that of Zhongmu No. 1, and whether the appearance of introns in these genes affects the original functions is a question that needs further research.In general, TFs always harbor some important conserved domains and motifs for their regulatory function . Throughc finger . When gec finger . Almost ZF-HD genes are differentially expressed in different tissues of different species, indicating that they have an important role in the development of plant tissues [4 plant Flaveria bidentis, and they are involved in a variety of biological processes, such as the response to abiotic stress [ZF-HD genes under alkaline stress has not been previously investigated. Therefore, both XinJiangDa Ye and Zhongmu No. 1 were treated with alkaline stress and samples were taken at 5 different time periods to explore the expression of MsZF-HD genes in response to alkaline stress. Many MsZF-HD genes could not be detected after the alkaline treatment, which may indicate that these genes are expressed only in specific environmental conditions or tissues. It was worth noting that the differentially expressed gene in the root tips under salt stress were not the same as those in the leaves under alkali stress. For example, MsZHD-Z5 was significantly up-regulated in the root tips under salt stress, while down regulated in the leaves under alkali stress. In conclusion, the functions of MsZF-HD gene are different under salt and alkali stress. Most MsZF-HD genes expressed the highest levels in the early stage of the alkaline stress treatment and stabilized after 12 h, reaching a very low level, thus indicating that the ZF-HD TFs respond to alkaline stress quickly and may be upstream of the signal transduction pathway. Constitutive overexpression of the AtMIF1 gene leads to significant developmental defects, including dwarfing, reduced apical dominance, altered flower morphology, reduced hypocotyl length, reduced root growth, and ectopic root hairs on hypocotyls and cotyledons [ZF-HD genes, MsMIF also included a few genes that could respond to alkaline stress, although there were certain differences. The MIF subfamily genes of alfalfa have a low expression level, and only two expressed genes (MsMIF-X2–1 and MsMIF-X3–1) showed a trend of initially increasing and then decreasing. tissues . ZF-HD Tc stress  and hormc stress . ZF-HD Tc stress , fruit dc stress , salt stc stress  and drouc stress . Howevertyledons . SimilarMsZF-HD genes in M. sativa L and then characterized these genes and explored their phylogenetic relationships. The expression analysis showed that most MsZF-HDs also respond to alkaline stress, in addition to drought, salt and ABA stress.In summary, this study identified 60 https://figshare.com). The A. thaliana genome and annotation information were downloaded from the TAIR database (https://www.arabidopsis.org/), and the 17 AtZF-HD protein sequences were downloaded from TFDB . The MsZF-HD genes were searched using 2 methods: First, the published AtZF-HD protein sequences were used as probes for a BLAST search against the proteome of alfalfa to obtain candidate ZF-HD family members. Second, the hidden Markov model (HMM) profiles of the ZF-HD_dimer domain (PF04770) were downloaded from the Pfam database (http://pfam.xfam.org/) to search for the ZF-HD_dimer domain from the alfalfa proteome using an e-value cut off of 1.0. Duplicate genes obtained by the two methods were removed to obtain the final MsZF-HD genes. The NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov) was utilized to analyze the domains of candidate ZF-HD proteins to ascertain the candidate genes for subsequent analysis. Finally, the nucleotide and deduced amino acid sequences of 60 MsZF-HD genes were confirmed for further analysis. The amino acid sequence lengths, molecular weights (MW) and pI values of all predicted MsZF-HD proteins were then determined by ExPASy (http://web.expasy.org/compute_pi/). All related information of MsZF-HD genes are listed in Table The genome and annotation information of alfalfa (XinJiangDa Ye and Zhongmu No. 1) were downloaded from the figshare data repository (Arabidopsis were used to construct phylogenetic trees by the maximum likelihood (ML) method with 1000 bootstrap replicates through MEGA X software. The parameters were set as follows: protein model: JTT + G + I; gap/missing date treatment: partial deletion; and site coverage cut off: 95%.To study the phylogenetic relationships of the different ZF-HDs, the full-length amino acid sequences of ZF-HD members of alfalfa and ZF-HD genes were predicted by MEME (https://meme-suite.org/meme/tools/meme). The optimized parameters of MEME were as follows: the optimum width of each motif ranged from 6 to 50, and the maximum number of motifs to find was 10. In addition, TBtools [http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict and analyze the cis-elements of 2000 bp upstream sequences of each MsZF-HD gene.Conserved motifs and domains of  TBtools  was usedMsZF-HD genes in alfalfa chromosomes, reference information for the alfalfa genome was obtained. The chromosomal distribution and synteny were analyzed using TBtools. The proteomes of M. sativa, A. thaliana, M. truncatula, and G. max were blasted to find duplicated genes between different species. Multiple collinear scanning toolkits (MCScanX) were used to analyze the replication events of the MsZF-HD genes. The syntenic relationship between the MsZF-HD genes and ZF-HD genes from selected plants was determined using Dual Systeny Plotter software (https://github.com/CJ-Chen/TBtools).To analyze the distribution of MsZF-HD genes, with the phylogenetic relationships as well as their subgenome location in XinJiangDa Ye  taken into account. Each gene name starts with an abbreviation for the species, e.g., Ms. for M. sativa L., followed by the name of the subfamily, e.g., ‘ZHD’ for ‘zinc finger motif-associated HD’ and ‘MIF’ for ‘mini zinc finger’. In addition, Z and X indicate which variety they represent. The MsZF-HD genes belonging to XinJiangDa Ye but on different homologous chromosomes were consecutively numbered and separated by a dash. Hence, the name of the gene with the ID MS.gene062215 is MsZHD-X1–1, indicating that it is a ZHD subfamily gene and the first homologous chromosome and first gene of XinJiangDa Ye.We considered a consistent naming pattern for all M. sativa (XinJiangDa Ye and Zhongmu No. 1) was employed for the alkaline treatment experiments. Uniform seedlings were transplanted into plastic cylindrical pots  containing vermiculite and perlite (1:1) under 16/8 h light/dark conditions, 30 °C/25 °C day/night temperatures and 55% relative humidity. The pots were placed in rectangular plastic trays . During plant growth, 1/5 Hoagland nutrient solution was applied to the plants once every 2 days. Two-month-old seedlings were utilized for the alkaline treatment. For alkaline stress, plants were treated with 250 mM NaHCO3 (pH = 8.5), and leaves of the seedlings were harvested at 0, 3, 6, 24, and 48 h after treatment. Immediately, the collected samples were frozen and stored at − 80 °C until use. For all the above samples, three biological replicates were employed for each sample.M. sativa transcriptome data under different abiotic stresses were downloaded from the NCBI SRA database . Different time points of three abiotic stresses, including 250 mM NaCl, 10 μM ABA, and 400 mM mannitol, were analyzed. The transcript abundance is expressed based on the fragments per kilobase of exon model per million mapped reads (FPKM) values. Heat maps for MsZF-HD genes that have positive FPKM values in at least one or more of the samples were generated, and the values are shown with log2 (1 + RPKM). We also performed RNA-seq to detect the expression levels of MsZF-HD genes under alkaline stress. Clean reads from 30 samples were mapped to the M. sativa genome sequencing using SamTool [ZF-HD genes were utilized to generate the heatmap and k-means clustering using R (software) [ SamTool . The FPKoftware) .GAPDH gene (accession: XM_003601780.1) was used as an internal reference gene. The relative gene expression levels were calculated according to the 2−∆∆C(t) method. SPSS 2.5 and Excel software were used for data analysis, and Duncan’s method was used to compare significant differences between treatments (p < 0.05), which are marked with different lowercase letters. All data were the mean ± standard error of 3 biological replicates with 3 technical replicates. All qRT-PCR validation primers used in the present study are listed in Table Total RNA was extracted from each sample using the Plant RNAprep Pure Kit . First-strand cDNA was synthesized from 1 μg of total RNA using a HiScript II Q Select reverse transcriptase kit  according to the manufacturer’s instruction. Gene-specific primers were designed using Primer Premier 5 software. In each reaction, the Additional file 1: Supplementary Table 1. One-to-one orthologous relationships between Medicago sativa L. and other three plant species. Supplementary Table 2. The FPKM value of MsZF-HD genes under different abiotic treatment.


Endometriosis is a common gynecological disease, affecting up to 10% of women of reproductive age and approximately 50% of women with infertility. Circular RNAs (circRNAs) have been shown to be involved in a number of diseases. Dysregulated expression of circRNAs in endometriosis has been reported, and circ_0000673 was significantly downregulated. However, the details of its role in the pathogenesis of endometriosis are still poorly understood. We investigated the location and effects of the downregulation of circ_0000673 in endometriosis. We demonstrated that knockdown of circ_0000673 significantly increased the proliferation and migration of eutopic and normal endometrial cells. Bioinformatics analysis predicted that circ_0000673 might act as a sponge for miR-616-3p. We found that the effect of circ_0000673 knockdown could be recovered by miR-616-3p inhibitor and enhanced by miR-616-3p mimics. qPCR and western blot assays showed that circ_0000673 knockdown could decrease the expression of PTEN and increase the expression of PI3K and p-AKT. PTEN was confirmed to be a target of miR-616-3p. These results demonstrated that the downregulation of circ_0000673 could promote the progression of endometriosis by inactivating PTEN via the deregulation of miR-616-3p. Endometriosis is a debilitating disorder that is caused by the presence of endometrial cells outside the uterine cavity. It affects up to 10% of women of reproductive age, affecting over 176 million women worldwide RSL1D1 gene and is 251 bp long. The RSL1D1 gene contributes to the cell cycle, cell proliferation, apoptosis, and metastasis, by downregulating the expression of the PTEN gene Circular RNAs (circRNAs) are a class of endogenous RNAs with a closed loop structure that makes them more stable than linear RNAs. circRNAs are highly abundant and specifically expressed in different tissues and developmental stages and have been found to participate in the regulation of biological and pathological processes such as cell proliferation, apoptosis, invasion, angiogenesis, and the epithelial-mesenchymal transition (EMT), by acting as microRNA (miRNA) sponges Using bioinformatics analysis, we found that circ-0000673 contains an miR-616-3p response element. miR-616-3p is reported to be a carcinogenic miRNA (onco-miRNA) in ovarian cancer, breast cancer, and gastric cancer, acting by promoting cell proliferation, reducing apoptosis, and promoting cell invasion and metastasis, the EMT, and other processes. Dysregulated miR-616-3p contributes to abnormal proliferation and apoptosis by targeting PTEN in hepatocellular carcinoma, renal tubular epithelial cells, and cardiomyocytes This study was approved and supervised by the Medical Ethics Committee of Xiangya Hospital, Central South University. Written informed consent was obtained from all subjects. Twenty patients with ovarian endometriomas at stage III-IV who provided both EU and EC and 20 women without endometriosis, as normal endometrial (NE) controls, were recruited during the proliferative phase. All subjects were 20-45 years old, with regular menstrual cycles, and had received steroid hormone treatment at least 3 months before specimen collection.2O2 was added to block endogenous peroxidase. After prehybridization, the hybridization solution with a digoxigenin (DIG) -labeled probe was added, and the sections were hybridized overnight at 42 °C. The sections were then incubated with mouse anti-digoxigenin-labeled peroxidase  at 37 °C for 50 min. After washing three times, freshly prepared FITC-TSA chromogenic reagent  was added to the reaction in the dark for 5 min at room temperature, and then the sections were incubated with DAPI for 5 min. Finally, the slices were sealed and observed using a Nikon Laser Scanning Confocal Microscope .The EU and EC tissues were embedded in paraffin and sliced. Slices were dewaxed in xylene, dehydrated in anhydrous ethanol, and then treated with protease K (20 µg/mL). Subsequently, 3% methanol-H2 at 37 °C. The isolated endometrial stromal cells were identified by staining with vimentin using immunofluorescent techniques as previously described The endometrial tissue was rinsed with Hanks' solution and cut into pieces in F12/DMEM culture medium, followed by collagenase digestion for 60 min and DNase I digestion for 30 min. After centrifugation, the cells were suspended in F12/DMEM with 10% fetal bovine serum and filtered through a 70-μm cell strainer to collect endometrial stromal cells. The endometrial stromal cells were inoculated in F12/DMEM with 10% fetal bovine serum in an incubator with 5% CO4 mL. Lentivirus vectors  targeting the junction region of hsa_circ_0000673 were constructed to knockdown the expression of hsa_circ_0000673 in endometrial stromal cells. All lentivirus vectors were transfected into eutopic and normal endometrial stromal cells with transfection enhancers. The sequences were as follows: LV-1, 5′-GTGGTTCTTGCAGATTATCTC-3′; LV-2, 5′-CTTGCAGATTATCTCCCTCCA-3′; LV-negative control (NC), 5′-TTCTCCGAACGTGTCACGT-3′. The transfection efficiency was confirmed using fluorescence microscopy after 24 h, and cells were harvested for further functional experiments after 48 h. Has-miR-616-3p mimic or inhibitor  was transfected using Lipofectamine 2000 reagents  according to the manufacturer's protocol. All experiments were performed three times in each assay.Primary eutopic and normal endometrial stromal cells were treated at the third passage, with a cell density of 1 × 10The CCK-8 assay was carried out using Cell Counting Kit-8  according to the manufacturer's protocol. Briefly, cells were seeded into 96-well plates and divided into four groups, with four duplicate wells in each group. After cell attachment, the lentivirus vectors were added, and the plates were incubated at 37 °C for 24 h. 10 μL CCK-8 reagent was added into each well, and the plates were incubated at 37 °C for 2 h. Cell proliferation was determined by testing the absorbance value at 450 nm using a spectrophotometer.Cells were placed in six-well plates with 1000 cells per well. LV-circ_0000673 or miR-616-3p mimic/inhibitor was transfected, and the cells were cultured for 2 weeks. After washing, the cells were successively treated with 4% paraformaldehyde and 0.1% crystal violet. The number of colonies in each well was counted under the microscope.Wound healing assays were performed to assess the migration capability of endometrial stroma cells after knockdown of circ_000673. Briefly, endometrial stromal cells were seeded in six-well plates, and LV-circ_0000673 or miR-616-3p mimic/inhibitor was transfected. Then, the monolayered cells were scraped with a micropipette tip to make a straight wound and cultured with serum-free medium for 24 h. The gap width at 0 and 24 h was recorded using microscopy.-ΔΔCt method.Cells were collected 72 h after transfection, and total RNA was extracted using TRIzol reagent. The concentration and purity of the RNA were measured using a NanoVue Plus spectrophotometer . The primers were listed in Table Endometrial cells were lysed in RIPA lysis buffer to extract total protein. Aliquots of 50 μg total protein were taken for 10% SDS-PAGE electrophoresis, transferred onto a 0.45-μm PVDF membrane, and blocked with 5% nonfat milk. Primary antibodies of PTEN , PI3K , p-AKT , and GAPDH  were added and incubated overnight at 4 °C. Then, after washing for three times, the secondary antibodies were added and incubated at room temperature for 1 h. Finally, enhanced chemiluminescence reagent  was added, and the reactivity was determined by an enhanced chemiluminescence detection system .Dual luciferase assays were performed to assess the binding between circ_0000673, PTEN, and miR-616-3p. The wild-type (WT) circ_0000673 and the PTEN 3′-UTR were inserted into pMIR-reporter plasmids , and the mutant (MUT) type was designed by mutating the binding site of the seed sequence. The WT and MUT luciferase reporter plasmids were co-transfected with miR-616-3p mimics or negative control into 293T cells. The relative luciferase activity was measured using luciferase reporter kits .t tests were performed for pairwise comparisons, and the one-way analysis of variance was used for multiple comparisons. All experiments were performed in triplicate, and a P value <0.05 was taken to indicate statistical significance. The statistical analysis was conducted using SPSS 25.0 and GraphPad Prism 9.0 .Data were expressed as mean ± standard deviation (SD). Two-tailed Student's in situ hybridization also showed that circ_0000673 was expressed at low levels or not expressed in EC, whereas it was upregulated in EU. It was mainly distributed in the cytoplasm (Figure qPCR confirmed that circ_0000673 was significantly downregulated in EC compared with EU Figure A. Fluorem Figure B.Endometrial stromal cells presented a fusiform cell morphology under light microscopy, and the positive expression of vimentin by immunofluorescence stain confirmed the presence of endometrial stromal cells Figure A. TransfcircRNAs have been known to be involved in regulating gene expression by serving as miRNA sponges. We predicted circRNA-miRNA interactions using the TargetScan To further clarify the mechanisms of circ_0000673/miR-616-3p with respect to endometrial cell proliferation and migration, cells were transfected with circ_0000673 lentivirus knockdown vectors and/or miR-616-3p mimics/inhibitors. Cells transfected with knockdown LV-circ_00007673 or miR-616-3p mimic showed significantly increased proliferation capacity, according to CCK-8 and colony formation assays Figure A, B, andmiRNAs are known to be important post-transcriptional repressors, acting by binding to the 3′-UTR. We conducted luciferase reporter assays to assess whether the expression of PTEN was directly regulated by miR-616-3p. miR-616-3p mimics significantly reduced the luciferase activity when co-transfected with PTEN-WT plasmids, whereas no effect on luciferase activity was observed when they were co-transfected with PTEN-MUT plasmids. These data confirmed that miR-616-3p targeted PTEN Figure .Endometriosis is an enigmatic disease, affecting mainly women of reproductive age and causing pain and reduced fertility. The treatments available are sub-optimal because of the uncertainty of the pathogenesis of endometriosis in situ hybridization, a finding that implied that it plays a role in ovarian endometriosis. Our findings indicate that loss of circ_0000673 could promote the proliferation and migration of endometrial cells. Recent studies have suggested that circATRNL1 promotes the EMT by regulating Yes-associated protein 1 The proliferation and migration of endometrial tissue outside of the uterus is a defining characteristic of endometriosis, although the details of the pathological progression remain largely unknown circRNAs have been known to play important roles in miRNA targeting. We found that circ_0000673 could bind to miR-616-3p, which may target PTEN. The levels of PTEN are reduced in endometriosis and associated with the stage of the condition The role of miRNAs is the degradation or inhibition of gene translation by binding to the 3′-UTR of the target gene. We found that PTEN was predicted to be the target of miR-616-3p and the direct binding relationship was confirmed by dual luciferase assays. PTEN is a tumor-suppressor gene with specific phosphatase activity and plays important roles in cell growth, proliferation, and migration via the PI3K-AKT pathway The dysregulation of circRNAs has been implicated in the pathology of several diseases, and decreases in the abundance of the circRNA circ_0000673 have been observed in endometriosis. Our study investigated the location and downregulation of circ_0000673 in tissues from patients with endometriosis, and the effects of its downregulation. We found the downregulation of circ_0000673 increased the expression of miR-616-3p and further blocked the target, PTEN, which then activated PI3K and p-AKT and promoted the proliferation and migration of endometrial stromal cells. Meanwhile, inhibiting miR-616-3p increased the expression of PTEN, and attenuated the cell proliferation and migration. The study revealed the roles of circRNAs in endometriosis, which may bring new approaches to exploring novel therapeutic targets for endometriosis.


The relationship between baseline fasting blood glucose (FBG) levels and 1-year stroke recurrence in non-diabetic patients with acute cerebral infarction (ACI) is unclear. We aimed to clarify this relationship in non-diabetic patients with ACI.Baseline FBG levels and related information of the patients were collected at admission and the events of stroke recurrence were followed up 1, 3, 6, and 12 months after the patients were discharged. Baseline FBG levels were analyzed as continuous variables and quartiles (Q1–Q4). Multivariate Cox regression models and a two-piecewise linear regression model were used to investigate the relationship and determine the threshold effect between baseline FBG levels and 1-year stroke recurrence in non-diabetic patients with ACI.P = 0.031]. Plotting hazard ratios over baseline FBG levels suggested a J-shaped relationship for 1-year stroke recurrence. Further analysis revealed that the nadir value of baseline FBG levels is 4.6 mmol/L. The relationship was more significant in patients with atrial fibrillation than in those without (P for interaction = 0.009).Overall, 1,634 non-diabetic patients with ACI were enrolled. After adjusting for potential confounding factors, the hazard is 2.24-fold higher in Q4 than those in Q2, being considered the reference in non-diabetic patients with ACI [hazard ratio (HR) = 2.24, 95%CI: 1.08–4.65, Lower and higher baseline FBG levels may lead to an increased risk of 1-year stroke recurrence in non-diabetic patients with ACI as shown by a J-shaped curve with a nadir value of 4.6 mmol/L. Acute cerebral infarction (ACI) is the most common type of stroke, accounting for higher than 70% of all strokes. It is now the second leading cause of death worldwide and the leading cause of death and disability-adjusted life-years in China –3.Previous studies showed that hyperglycemia occurs in 30–40% of patients with acute ischemic stroke. Most of these patients do not have a known history of diabetes . AlthougIn contrast, other studies have revealed the relationship between hyperglycemia and functional outcome in both diabetic and non-diabetic patients with ACI , 11. HowTo date, few studies have examined the relationship between baseline FBG and 1-year stroke recurrence in non-diabetic patients with ACI, especially in China. Thus in the present study, we collected the baseline FBG levels and related clinical data from non-diabetic patients with ACI in Xi'an Stroke Registry Study in China, hoping to investigate the relationship between baseline FBG levels and 1-year stroke recurrence after ACI onset and to provide a more accurate reference range of baseline FBG for preventing and treating 1-year stroke recurrence in non-diabetic patients with ACI as well as a theoretical basis for individual prevention and treatment.n = 629), diabetes  at admission (n = 329) or discharge (n = 243), missing baseline FBG value (n = 93) and lost during the 1-year follow-up (n = 189). ACI was diagnosed according to the WHO criteria and confirmed by brain MRI or CT  score ≤ 4 was classified as a mild deficit, 5–14 as a moderate deficit, and >14 as a severe deficit. The observed endpoint event in this study was stroke recurrence, which was defined as new acute stroke events  during the 1-year follow-up  and Free Statistics software versions 1.1.Continuous variables are presented as Mean ± SD and categorical variables are presented as percentages (%). A one-way ANOVA was used for normally distributed continuous variables to assess differences between groups; a chi-square test and trend test were used for categorical variables. When the sample did not satisfy a normal distribution, a Mann-Whitney U test was used. Multiple groups were compared using the Kruskal-Wallis rank-sum test. A Fisher's exact test was used when the theoretical frequency was <10. Confounding factors were controlled by multivariable-adjusted Cox regression models . The covAt the end of the one-year follow-up, 1,634 patients were not lost to follow-up while 189 were lost. By comparing the clinical characteristics of the two groups with or without patients lost to follow-up, only age, education level, and medical insurance type differed between the two groups and there was no statistical difference in other variables . This reA total of 1,634 eligible subjects  were included in our study. The mean age was 64.5 ± 12.7 years. The mean baseline FBG level was 5.2 ± 1.1 mmol/L. The baseline demographic, clinical, and biochemical characteristics of the baseline FBG level quartile (Q1–Q4) groups were compared . The patP < 0.001; model I: HR = 1.31, 95%CI:1.17–1.47, P < 0.001). After adjusting for potential confounding factors, higher baseline FBG levels were still associated with a higher stroke recurrence in model II . Then, we analyzed the baseline FBG level as categorical variable quartiles (Q1–Q4). After adjusting for potential confounding factors, the hazard is 2.24-fold higher in Q4 than those in Q2, which was considered the reference in non-diabetic patients with ACI . In addition, the Kaplan-Meier curve analyses also showed that the 1-year recurrence rate of stroke in Q4 was significantly higher (y higher .P = 0.029), whereas to the right of the cut-off value, the risk of 1-year stroke recurrence significantly increased . The above results indicated that lower and higher baseline FBG levels were associated with high 1-year stroke recurrence . Although the differences were not significant in the trend test (P = 0.068), the risk of stroke recurrence in Q3  and Q4  were higher than that in Q2. Moreover, the baseline FBG level in Q4 was associated with 1-year stroke recurrence in patients with hypertension  but not significant in interactive analyses (P for interaction = 0.25). Similar results were found for patients without pneumonia  and without previous stroke  but not significant in interactive analyses.To assess whether the relationship between baseline FBG levels and 1-year stroke recurrence among different subgroups is consistent, we performed stratified analyses and interactive analyses . The datIn this multicenter cohort study, we found that either lower or higher levels of baseline FBG were associated with an elevated risk of 1-year stroke recurrence, and the relationship between them presented as a J-shaped curve with a baseline FBG nadir value of 4.6 mmol/L in non-diabetic patients with ACI in Xi'an area.Previous studies have reported that multiple biomarkers, such as non-HDL cholesterol , VitaminP < 0.05) than that in other groups. After adjusting for potential confounding factors, the hazard is 2.24-fold higher in Q4 than those in Q2, being considered as the reference in non-diabetic patients with ACI. These results imply that higher levels of baseline FBG were associated with an elevated risk of 1-year stroke recurrence in non-diabetic patients with ACI. Clinicians should be concerned with rapidly elevated baseline FBG levels since they may be associated with an even greater risk of 1-year stroke recurrence.Literature reviews found that most previous studies had pooled patients with and without diabetes together. Thus, the results could not completely rule out the effect of diabetes, even though the confounding factors were adjusted for. Although several investigations focused on the relationship between baseline FBG levels and short-term death, disability, or poor outcome of non-diabetic patients with stroke , 11, 28,Furthermore, the relationship and optimal range of baseline FBG levels with respect to 1-year stroke recurrence had not been elucidated so far. To clarify this relationship, our study systematically analyzed the threshold effect and dose-response relationship between baseline FBG levels and 1-year stroke recurrence in a large-scale cohort study. The results confirmed a non-linear relationship. After adjusting for potential confounders, a J-shaped curve between baseline FBG levels and 1-year stroke recurrence was observed in non-diabetic patients with ACI . The optOur result was inconsistent with those in previous correlational studies. Ntaios found a J-shaped curve between blood glucose levels and 12-month functional outcome in patients with ischemic stroke, and initial serum glucose values between 3.7 and 7.3 mmol/L were found to be associated with a favorable outcome . StaszewP = 0.013). The relationship between hypertension and the increased recurrence of stroke has been well demonstrated (P = 0.068), possibly due to the smaller number of patients with atrial fibrillation after stratification. Previous studies showed that the relationship between atrial fibrillation and increased stroke recurrence remains controversial  in Q4  the four hospitals included in this study were not randomly selected, thus there was potential for selection bias; (2) all the selected centers were tertiary-grade A hospitals that may not represent the status quo of non-diabetic patients with ACI in community hospitals; (3) our stroke database did not have data on the concentration of FBG changes over time in this registration-based study; (4) glycated hemoglobin test was not included in this study, thus the patients with mild diabetes being undiagnosed previously could not be excluded. We will further improve our database in the follow-up study.Our study reveals a J-shaped curve between baseline FBG level and 1-year stroke recurrence with a nadir value of 4.6 mmol/L, indicating that both the lower and higher baseline FBG levels may lead to an increased risk of 1-year stroke recurrence in non-diabetic patients with ACI. Our findings may help physicians to assess the 1-year stroke recurrence in non-diabetic patients with ACI in the acute phase.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee at Xi'an No.1 Hospital. The patients/participants provided their written informed consent to participate in this study.SW and YT had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. ZL and SW planned and designed the study. ZL contributed to the data cleaning and statistical analysis. ZL, XL, and WL wrote the manuscript. QL, PL, JW, YL, QC, YW, and CS contributed to follow-up patients and recorded the data at each stage. FW, YS, QW, and GL revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.The study was supported by the Science and Technology Program of Shaanxi Province (Grant Nos. 2017SF163 and 2021SF-333), the Science and Technology Plan Major Project of Xi'an city [Grant No. 201805104YX12SF38(2)], the Science and Technology Plan Project of Xi'an city [Grant No. 20YXYJ0008(1)], and the Scientific Research Project of the Xi'an Health Commission . The funders had no role in the design and analysis of this trial.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.